B.D. Mariani

Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes

The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting ‘karyomap’, unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.

Malignant precursor cells pre-exist in human breast DCIS and require autophagy for survival.

Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patients breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patients original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from the organ culture, and abrogated xenograft tumor formation. Conclusions Cytogenetically abnormal spheroid forming, tumorigenic, and invasive neoplastic epithelial cells pre-exist in human DCIS and require cellular autophagy for survival.

Human herpesvirus 6 rhombencephalitis in immunocompetent children.

This article describes the clinical presentation, diagnostic workup, and neurologic outcome of 3 immunocompetent pediatric patients diagnosed with human herpesvirus 6 (HHV6) rhombencephalitis. Presentation of HHV6 rhombencephalitis included new onset seizures, ataxia, encephalopathy, and opsoclonus-myoclonus. Neurologic examination revealed cranial neuropathies, cerebellar dysfunction, and extremity weakness. Magnetic resonance imaging abnormalities located in the cerebellum, basal ganglia/thalamus, and cerebral hemispheres were detected in 2 patients. Diagnosis of HHV6 encephalitis was made by real-time and nested polymerase chain reaction of serum and cerebrospinal fluid. The HHV6 variant A was detected in 2 patients by sequence analysis, and HHV6 protein was detected by immunomicroscopy in a patient who underwent biopsy secondary to progressive clinical and neuroradiographic findings. Therapy with intravenous ganciclovir did not correlate with resolution of neurologic symptoms, despite eventual non-detectable HHV6. Human herpesvirus 6 should be considered in the differential diagnosis of unexplained cases of rhombencephalitis in immunocompetent children. Features may be rapidly progressive and include profound encephalopathy, seizures, ataxia, and opsoclonus-myoclonus.

The accuracy of chromosomal microarray testing for identification of embryonic mosaicism in human blastocysts

BackgroundMost previous studies of chromosomal mosaicism in IVF embryos were performed by fluorescence in situ hybridization (FISH) methods. While there are reports implicating chromosome aneuploidy in implantation failure following transfer and pregnancy loss by spontaneous miscarriage, the significance of mosaicism for the developmental potential of growing embryos is unknown. However, the low prevalence of chromosomal mosaicism in chorionic villus sampling and amniotic fluid specimens suggests the presence of selection against mosaic embryos for implantation and early pregnancy. The absence of evidence for selective allocation of abnormal cells to the trophectoderm (TE) of mosaic blastocysts permits these cells to be a good proxy for embryonic mosaicism detection by chromosomal microarrays (CMA). The purpose of this study was to establish the limits of detection and the prevalence of chromosome mosaicism in day 5/6 human embryos using CMA with TE biopsies.ResultsFrom reconstitution experiments we established log2 ratio thresholds for mosaicism detection. These studies indicated that chromosomal mosaicism at levels as low as between 25-37% can be consistently identified. Follow-up studies by FISH on non-transferred abnormal embryos confirmed the diagnostic accuracy of CMA testing. The number of cells in a TE biopsy can influence mosaicism detection.ConclusionsChromosomal microarrays can detect mosaicism in TE biopsies when present at levels as low as between 25-37% and the prevalence of day 5/6 blastocysts which were mosaic and had no other abnormalities reached 15% among a cohort of 551 embryos examined. Validated protocols for establishing detection thresholds for mosaicism are important to reduce the likelihood of transferring abnormal embryos.

Acute and chronic magnetic resonance imaging of human herpesvirus-6 associated encephalitis

Human herpesvirus-6 (HHV-6) has been associated with central nervous system encephalitis in immunocompetent children. To determine acute and chronic changes on magnetic resonance imaging (MRI) of a series of consecutive pediatric immunocompetent patients diagnosed with HHV-6 associated encephalitis. A retrospective review of acute and chronic MRI findings of immunocompetent children diagnosed with HHV-6 associated encephalitis from 2001-2008 was performed. Diagnosis was established by real time polymerase chain reaction using HHV-6 U77 helicase gene-specific primers on cerebrospinal fluid during the acute phase of illness, excluding other known causes of encephalitis. Seven patients (four girls, ages 9 months-7 years) were diagnosed with HHV-6 associated encephalitis from 2001-2008. MRI abnormalities were present in six of seven patients. Three had bithalamic signal abnormalities on T2 weighted sequences that mimicked metabolic or demyelinating disease. All patients with MRI abnormalities had diffuse signal changes in the brainstem (n =5 ) and/or cerebellum (n =4 ). One patient with diffuse abnormalities in cortical grey and white matter, hippocampus, and cerebellum showed complete resolution of MRI findings after 1 month. Five patients had extensive necrosis of the acutely affected regions on follow up MRI 3 to 16 months post diagnosis. HHV-6 associated encephalitis can occur in immunocompetent children with a myriad of acute and chronic necrotizing changes on MRI. MRI abnormalities were seen predominantly in the thalami, brainstem, and cerebellum; and may be reflective of anatomical viral tropism. HHV-6 should be considered in the differential diagnosis of unexplained MRI changes in patients with encephalitis.

Bioanalytical PCR: ready for Prime time?: PCR in Bioanalysis edited by S.J. Meltzer

Humana Press 1997. US

Recombination mapping: a universal molecular karyotyping method for preimplantation genetic diagnosis of inherited disease

69.50 (xi + 281 pages)ISBN 0-896-03497-6The ability to amplify specific nucleic acid sequences from biological sources that have been previously beyond the reach of molecular analysis has dramatically expanded our investigative and analytical capabilities. During 15 years of tinkering with PCR, the time is well overdue for defining exactly how the technology can be used as an analytical tool. The first step in that process is to identify key areas in which good ground work has already been done and where innovation can still be applied. To this end, numerous authors and editors have tried to compile examples of the best ways to use PCR in recent years. But with the plethora of PCR methodology books available today, it can be time consuming to distinguish the good works from the excellent ones. Because PCR in Bioanalysis is excellent, the serious PCR-based investigator can be saved valuable time by turning straight to this volume. It incorporates a wide range of applications and approaches in an accessible and contemporary volume. Despite the fact that the collection of 24 chapters covers a wide range of interests, the diversity is not a distraction because all the presentations have a bioanalytical focus. The first chapter, which presents an overview of the theory, practice and performance of PCR, is one of the best that I have read, and it has become required reading by my laboratory personnel, regardless of their experience. I was grateful that this single chapter distills the essential aspects of performing analytical PCR, and other related amplification technologies, in the minimum of space and with the utmost clarity.The remainder of the volume is conceptually divided into general topics that cover: (1) PCR applications in animal and agricultural science, including transgenic mice and transgenic plant molecular biology; and (2) PCR detection and diagnosis of human genetic and infectious diseases, including a small collection of papers that describe innovative approaches to analyzing genetic heterogeneity in cancerous tissue and to finding termination mutations in large, complex genes.In the former section, several papers focus on the use of PCR for detecting and differentiating between strains of viruses that infect bovine and porcine species. This section addresses issues of disease transmission and specimen processing; a focus that is equally useful to clinical virologists who study disease transmission and diagnosis in humans. Bovine immunodeficiency-like virus (BIV) has been known for 25 years; the hard lessons that have been learned from inconclusive serological data and the difficulty of virus isolation in that field can be very instructive today for those working on HIV and who are turning to PCR for significantly improved diagnostic power, sensitivity and resolution.The most highly refined reports in the volume are those that are directed toward the diagnosis of infection in human disease, and which are readily implementable for fieldwork. The editor did an excellent job of choosing among many possible areas of infectious disease to arrive at an instructive sampling of papers that focus on those pathogens (1) that are difficult, or impossible, to cultivate; (2) that exist as low level but transmissible asymptomatic infections; and (3) that require rapid diagnosis for the making of critical therapeutic decisions. In the paper that addresses the sexually transmitted diseases, attention is paid to the fact that improved sensitivity allows the testing of alternative, less invasive specimen types, thus increasing patient compliance, an issue that is not normally considered in methodology textbooks. The chapter on tuberculosis diagnostics contains an in-depth discussion of the potential savings in health-care costs, not only from one molecular testing scheme but also from three alternative approaches, extending the discussion of the impact of PCR bioanalysis into the realm of patient management and the improvement of health care. Other strong points in this category are two reports that use innovative non-isotopic approaches to focus on the devastating worldwide problem of enterotoxigenic Escherichia coli, and the difficulty that is encountered when attempting to type closely related bacterial strains. For Legionella infection, where typing is not an issue, a PCR strategy is presented that covers all 16 species, thus ensuring that a subtle, but important, diagnosis is not missed. A creative methodology is also presented that allows for more than just the detection of HIV RNA – it also allows the discrimination of the RNA subspecies, an approach that is valuable for investigating the progression of the viral life cycle and the progressive stages on the infection.One strength of this volume is that it addresses the traditional weaknesses of PCR. One of the shortcomings of PCR is that it is primarily, by nature, a sequence-dependent technique and is thus not particularly quantitative in its generic form. Although the power of PCR in mutational analysis has been well established for a few, well-defined, human genetic lesions, the challenge now is to extend its analytical capabilities of PCR to the detection of mutations in large genes or in genes that have multiple mutations that cover a wide range of sequence information. In this context, several laboratories have combined PCR with other sequence-independent molecular methods to extend the analytical power of the assay. This could lead to the analysis of additional, medically important, genes that have evaded a comprehensive analysis to date, such as the adenomatous polyposis coli (APC) gene. With regard to the quantitative use of PCR, several approaches are presented in detail that demonstrate how a quantitative component can be incorporated into a PCR analysis, whether by using an endogenous or exogenous template or an RNA or DNA source. Enough detail is presented for even the mildly intrepid bench worker to customize internal control templates for their specific targets. This ability to achieve quantitative PCR will be a key issue as the assay gains acceptance as a bioanalytical tool.Will PCR be an incisive bioanalytical tool? This book does not necessarily make it so or guarantee its success. The success of PCR as a bioanalytical tool will be up to those in the field who will have to use the tool correctly and with regard to its highly sensitive nature. This book only presents some useful, contemporary examples of what can be done if it is done correctly; it points the way for investigators to apply the technology analytically, if attention is paid to the parameters, the limits and to the controls, internal or otherwise.